Activation of Chloride Channels with the Anti-Parasitic Agent Ivermectin Induces Membrane Hyperpolarization and Cell Death in Leukemia Cells.

Abstract Abstract 410 FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. To identify such compounds, we compiled a library of known drugs with high maximal tolerated doses and well-known toxicity profiles. We screened this library in a dose-response manner in 4 leukemia cell lines to identify cytotoxic compounds as measured by the MTS assay. From these screens, we identified the anti-parasitic agent ivermectin (IVM) that induced cell death at low micromolar concentrations in all four leukemia lines tested. IVM is a derivative of avermecin B1 and licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis). To evaluate the effects of IVM as a potential anti-cancer agent, leukemia and myeloma (n = 9) cell lines were treated with increasing concentrations of IVM. 72 hours after incubation, cell viability was determined by the MTS assay. IVM decreased the viability of 3/5 leukemia cell lines with an LD50 < 5uM and all other tested malignant cell lines with an LD50 < 10uM. Cell death was confirmed by Trypan blue staining and Annexin V staining. In clonogenic growth assays, 6uM IVM reduced clonogenic growth by ≥ 40% in 3/6 primary AML samples, but <15% in 3/3 samples of normal hematopoietic mononuclear cells. Given the effects of IVM in cell lines, we evaluated the drug in mouse models of leukemia. Here, K562, OCI-AML2, and MDAY-D2 leukemia cells were implanted subcutaneously into the flanks of sublethally irradiated NOD/SCID mice. One week after implantation, when the tumors were palpable, mice were treated with IVM at 3mg/kg/day by oral gavage or buffer control. Compared to control, IVM decreased tumor volume and weight in all 3 xenograft models by up to 72% without observable toxicity. Of note, a dose of 3mg/kg in mice translates to a dose of 0.24 mg/kg in humans based on scaling of body weight and surface area. This dose is readily achievable in human as patients routinely receive 0.2mg/kg for the treatment of parasitic disease and overdoses of up to 6g were not toxic. As an anti-parasitic, IVM binds and activates chloride ion channels, so we tested the effects of IVM on chloride flux in leukemia cells. OCI AML2 cells were treated with increasing concentrations of IVM and changes in intracellular chloride were measured using the fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium and flow cytometry. IVM increased intracellular chloride ion concentrations within 30 minutes of treatment. Chloride influx was accompanied by plasma membrane hyperpolarization within 1 hr of treatment, but no change in mitochondrial membrane potential was noted up to 24 hours after treatment. Plasma membrane hyperpolarization was dependent on chloride influx, as treatment with IVM in chloride-free media did not induce membrane hyperpolarization. Alternations in intracellular chloride and membrane hyperpolarization can lead to increased reactive oxygen species (ROS) generation. Therefore, we measured changes in ROS after treatment with IVM. IVM (6uM) increased ROS generation in OCI-AML2 cells up to 2 ± 0.2 fold within 4 hours of treatment. Increased ROS generation appeared functionally important for IVM-induced cell death as pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) inhibited IVM-induced cell death. Further supporting a mechanism of cell death related to increased ROS, IVM treatment increased expression of STAT1, IFIT3, OAS1, and TRIM22, members of the STAT1 signaling pathway that are known to be upregulated upon ROS generation. Cytarabine and daunorubicin are used in the treatment of AML and increase ROS production through mechanisms related to DNA damage. Therefore, we evaluated the combination of IVM and cytarabine and daunorubicin. By isobologram analysis, IVM synergized with cyatarabine (CI=0.51, 0.58, 0.65 at ED25, ED50, ED75, respectively) and daunorubicin (CI=0.48, 0.51, 0.54 at ED25, ED50, ED75, respectively). Thus, in summary, IVM activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased ROS generation. In addition, it demonstrated preclinical activity in this disease at pharmacologically achievable concentrations. Therefore, IVM could be rapidly repurposed for the treatment of leukemia and highlights a potential new therapeutic strategy for this disease. Disclosures: Off Label Use: Ivermectin is an antiparasitic agent, licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis).

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The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells

Blood ◽

10.1182/blood-2010-01-262675 ◽

2010 ◽

Vol 116 (18) ◽

pp. 3593-3603 ◽

Cited By ~ 53

Author(s):

Sumaiya Sharmeen ◽

Marko Skrtic ◽

Mahadeo A. Sukhai ◽

Rose Hurren ◽

Marcela Gronda ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Leukemia Cell ◽

Reactive Oxygen Species Generation ◽

Reactive Oxygen ◽

Chloride Ion ◽

Leukemia Cells ◽

Parasitic Infections ◽

Oxygen Species ◽

Membrane Hyperpolarization

Abstract To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

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Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms22084265 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4265

Author(s):

Jang Mi Han ◽

Hong Lae Kim ◽

Hye Jin Jung

Keyword(s):

Signaling Pathways ◽

Cell Lines ◽

White Blood Cells ◽

Leukemia Cell ◽

Leukemia Cells ◽

Ros Generation ◽

Anticancer Activities ◽

Nuclear Condensation ◽

Leukemia Cell Lines ◽

Antileukemic Effect

Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.

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Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽

10.1182/blood.v120.21.3571.3571 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3571-3571

Author(s):

Matthew F. Clarkson ◽

Aru Narendran ◽

Randal N. Johnston

Keyword(s):

Cell Death ◽

High Risk ◽

Cell Viability ◽

Cell Lines ◽

Leukemia Cell ◽

Treatment Strategies ◽

Leukemia Cells ◽

Western Blots ◽

Pediatric Leukemia ◽

Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

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Uncovering Cardioprotective Strategies For Anthracycline Therapy By Analysis Of Redox and Apoptotic Effects

Blood ◽

10.1182/blood.v122.21.1293.1293 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1293-1293

Author(s):

Daniela E. Egas Bejar ◽

Joy M. Fulbright ◽

Fernando F. Corrales-Medina ◽

Mary E. Irwin ◽

Blake Johnson ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Vitamin E ◽

Acute Leukemia ◽

Cell Lines ◽

Leukemia Cell ◽

Cell Types ◽

Leukemia Cells ◽

Soluble Form ◽

Leukemia Cell Lines

Abstract Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants. Disclosures: No relevant conflicts of interest to declare.

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Endoplasmic Reticulum Stress Signaling Comprises a G9a Inhibitor Tolerance Pathway and PERK Inhibition Increases Anti-Leukemia Activity of G9a Inhibitor in Leukemia Cells and Leukemia Stem-like Cells

Blood ◽

10.1182/blood-2018-99-115814 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1360-1360

Author(s):

Jieun Jang ◽

Ju-In Eom ◽

Hoi-kyung Jeung ◽

So-Young Seol ◽

Haerim Chung ◽

...

Keyword(s):

Cell Death ◽

Er Stress ◽

Cell Lines ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Apoptotic Cell Death ◽

Leukemia Cells ◽

Dependent Manner ◽

Eif2α Phosphorylation ◽

Kg1 Cells

Abstract Background: Histone methyltransferase (HMTase) G9a regulates the transcription of multiple genes by primarily catalyzing dimethylation of histone H3 lysine 9 (H3K9me2), as well as several non-histone lysine sites. Recently, pharmacological and genetic targeting of the G9a was shown to be efficient in slowing down acute myeloid leukemia (AML) cell proliferation in a mouse model and human AML cell lines thus making this HMTase potential target for epigenetic therapy of AML. Activation of adaptive mechanisms to drug plays a crucial role in drug resistance and relapse by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The tolerance mechanism to HMTase regulation in leukemia cell is unclear yet. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Recent previous studies showed that pro-survival ER stress is induced in cancer cells and contributes to development of drug resistance. Methods: We investigated the levels of apoptosis and ER stress by G9a inhibitor BIX-01294 in leukemia cell lines. U937, cytarabine-resistant U937 (U937/AR) and KG1 were used. U937/AR cell line was established in our laboratory by exposing parental U937 cells to stepwise increasing concentrations of cytarabine. Results: We initially examined the expression of G9a in leukemia cell lines and the primary AML cells obtained from a patient at the different time point. In U937/AR cells and primary AML cells obtained at relapse, G9a expression was increased compare to that in U937 cells and primary AML cells obtained at diagnosis, respectively. G9a expression was also increased in KG1 cells. In both of U937 and U937/AR, apoptotic cell death was induced by BIX-01294 in a dose-dependent manner. In contrast, apoptotic cell death was minimal in KG1 cells which are enriched in cells expressing a leukemia stem cell phenotype (CD34+CD38-). To address the activation of ER stress response by BIX-01294 in leukemia cells, we examined the effect of BIX-01294 treatment on PERK and eIF2α protein expression and phosphorylation levels. We found that treatment of U937, U937/AR, KG1 cells with 3μM of BIX-01294 for 24h caused an upregulation of phosphorylated PERK and eIF2α. The upregulation of PERK phosphorylation was associated with a decrease in PERK protein levels after treatment. To further address the role of the PERK-eIF2α phosphorylation in BIX-01294 sensitivity, we examined whether PERK inhibition using small interfering RNA (siRNA) or specific inhibitor could sensitize cells to BIX-01294-mediated death. The siRNA against PERK effectively inhibited BIX-01294-mediated phosphorylation of PERK and eIF2α in U937 and U937/AR cells. The addition of PERK siRNA led to a significant increase in the extent of BIX-01294-induced apoptotic cell death in U937 (P = 0.0003) and U937/AR (P < 0.0001) as compared with that of BIX-01294 treatment alone. PERK inhibitor GSK260641 significantly increased BIX-01294-induced apoptotic cell death in U937 (P < 0.0001) and U937/AR (P = 0.006) cells. To our surprise, addition of PERK siRNA or GSK260641 increased the sensitivity of KG1 cells to BIX-01294-mediated death in a dose-dependent manner (P = 0.0003 for siRNA, P = 0.0053 for GSK260641). Conclusion: These data demonstrated that PERK-eIF2α activation has a pro-survival function to G9a inhibitor in leukemia cells and mediates resistance of AML stem cells to G9a inhibitor treatment. The PERK-eIF2α phosphorylation arm may represent a suitable target for combating resistance to G9a inhibitor in AML. The mechanisms underlying the increased sensitivity of AML cells with PERK inhibition to G9a inhibitor are unclear at present and are needed to define in further studies. Disclosures No relevant conflicts of interest to declare.

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Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL

Blood ◽

10.1182/blood.v87.9.3837.bloodjournal8793837 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3837-3843 ◽

Cited By ~ 96

Author(s):

A Benito ◽

M Silva ◽

D Grillot ◽

G Nunez ◽

JL Fernandez-Luna

Keyword(s):

Cell Death ◽

Cell Lines ◽

Leukemia Cell ◽

K562 Cells ◽

Erythroid Differentiation ◽

Leukemia Cells ◽

Human Leukemia ◽

Human Leukemia Cells ◽

Leukemia Cell Lines ◽

Wide Range

The induction of tumor cell differentiation represents an attractive strategy for the treatment of a wide range of malignancies. Differentiation of HL-60 promyelocytic leukemia cells towards neutrophils or monocytes has been shown to induce apoptotic cell death, which is inhibited by bcl-2 over-expression. However, the role of the bcl-2 gene family during erythroid differentiation of human leukemia cells remains unknown. We found that human erythroleukemia (HEL) and K562, two leukemia cell lines that undergo erythroid differentiation do not express Bcl-2, but express Bcl-XL, a related protein that functions as an inhibitor of apoptosis. Differentiation of HEL or K562 cells with inducers of erythroid differentiation (hemin, retinoic acid, or transforming growth factor-beta) was accompanied by progressive cell death and degradation of genomic DNA into oligonucleosomal fragments. The loss of cellular viability was associated with downregulation of bcl-xL mRNA and protein. In contrast, the levels of Bax, another Bcl-2 family member implicated in apoptosis remained unaltered. Constitutive expression of Bcl-XL by gene transfer inhibited apoptosis triggered by erythroid differentiation of HEL K562 cells. Yet, Bcl-XL did not alter the expression of epsilon-globin, which is induced during erythoid differentiation of HEL and K562 cells, arguing that apoptosis and differentiation can be uncoupled by Bcl-XL. These results indicate that Bcl-XL acts as an antiapoptosis protein in leukemia cells that undergo erythroid differentiation and that downregulation of bcl-x is a component of the apoptotic response that is coupled to differentiation in human leukemia cells.

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Targeting Mutant p53 in Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.903.903 ◽

2015 ◽

Vol 126 (23) ◽

pp. 903-903

Author(s):

Salih Demir ◽

Galina Selivanova ◽

Eugen Tausch ◽

Lisa Wiesmüller ◽

Stephan Stilgenbauer ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Cell Lines ◽

Therapeutic Intervention ◽

High Performance ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Therapy Resistance ◽

Leukemia Cells

Abstract Mutations of the tumor suppressor gene TP53 have been described to be associated with aggressive disease and inferior prognosis in different types of cancer, including hematological malignancies. In acute lymphoblastic leukemia (ALL), TP53 alterations are infrequently found at diagnosis but have recently been described in about 12% of patients at relapse. This suggests an association with therapy resistance in high risk/relapsed ALL and patients with TP53 mutated ALL have in fact an inferior outcome. Small molecule compounds targeting mutated TP53 such as APR-246, initially described as PRIMA-1MET (p53-dependent reactivation and induction of massive apoptosis) leading to apoptosis induction have shown activity in several types of malignancies with mutated TP53. In ALL, however, mutant TP53 has so far not been addressed as a target for therapeutic intervention. In this study, we investigated a large cohort of patient-derived pediatric B cell precursor (BCP)-ALL primograft samples to identify cases with mutated TP53. Further, we analyzed the effects of APR-246 and evaluated its activity on BCP-ALL cell lines and primografts with mutated (mut) orwild type (wt) TP53. Altogether, 62 BCP-ALL primograft samples established from patients at diagnosis (n=53) or relapse (n=9) by transplantation of primary ALL cells onto NOD/SCID mice were screened for TP53 mutations by denaturating high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of exons 4 to 10 to confirm detected mutations. We identified 4 cases with TP53 mut, 3 obtained from diagnosis (5.6%) and one at relapse (11.1%), corresponding to frequencies described in clinical studies. Mutated cases were further analyzed by fluorescence in situ hybridization (FISH), revealing a 17p deletion in one TP53 mut sample. Similarly, we analyzed 6 BCP-ALL cell lines and identified 2 TP53 mut and 4 TP53 wt lines. Exposure of BCP-ALL primograft (TP53 mut n=4, TP53 wt n=4) and cell line (TP53 mut n=2, TP53 wt n=4) samples to the DNA damaging agent doxorubicin showed, as expected, resistance of TP53 mut leukemia cells for cell death induction, reflected by significantly higher half maximal inhibitory concentrations (IC50; TP53 mut 49 and 143 ng/ml, TP53 wt mean 12 ng/ml) and lower induction of cell death (TP53 mut 16 to 23%, TP53 wt 10 to 60%) in TP53 mut ALL, corroborating the tumor-suppressive function of p53 in ALL. We then investigated the sensitivity of BCP-ALL cell lines for cell death induction by APR-246 (kindly provided by Aprea, Stockholm, Sweden). We observed high sensitivity for APR-246 in TP53 mut (IC50: 5 µM for both cell lines) as compared to TP53 wt ALL (mean IC50: 58 µM). DNA fragmentation and Annexin-V/propidium-iodide (PI) positivity revealed apoptosis as mechanism of APR-246 mediated cell death. Reactive oxygen species (ROS) have recently been described to mediate APR-246 induced cell death in multiple myeloma cells. Therefore, we investigated ROS levels by detection of oxidation-specific fluorescence of dichlorodihydrofluorescein diacetate (DCFDA) in ALL cells. Interestingly, ROS quenching by N-acetyl cysteine abolished induction of cell death in TP53 mut but not TP53 wt ALL cells indicating ROS as a mediator of APR-246 induced cell death in TP53 mut ALL. Furthermore, we addressed p53 activation in response to APR-246 by assessing phosphorylation of p53 (p53pSer15) using phosphoflow cytometry. Most interestingly, APR-246 led to 6-fold increased p53pSer15 levels in TP53 mut compared to no activation in TP53 wt leukemia cells, indicating restoration of p53function upon APR-246treatment in BCP-ALL. Based on these findings, we addressed the effectivity of APR-246on primary, patient-derived primografts and compared sensitivities for cell death induction in TP53 mut (n=4) and TP53 wt (n=4) samples. Importantly, the pattern of responsiveness of TP53 mut ALL was also identified in TP53 mut patient-derived ALL samples with induction of significantly higher cell death rates in TP53 mut ALL (TP53 mut 48%, TP53 wt 18%, 5 µM APR-246, 24 h). Taken together, we showed that TP53 mut BCP-ALL can be targeted by APR-246 leading to re-activation of p53, induction of ROS dependent apoptosis and effective leukemia cell killing. Thus, targeting and re-activation of mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL. Disclosures Selivanova: Aprea: Patents & Royalties: APR-246. Tausch:Gilead: Other: Travel support. Stilgenbauer:Gilead: Honoraria, Research Funding.

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Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽

10.1371/journal.pone.0256708 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256708

Author(s):

Christopher von Beek ◽

Linnéa Alriksson ◽

Josefine Palle ◽

Ann-Marie Gustafson ◽

Mirjana Grujic ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Tumor Formation ◽

Leukemia Cells ◽

Human Leukemia ◽

Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

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The Anti-Malarial Mefloquine Demonstrates Preclinical Activity In Leukemia and Myeloma, and Is Dependent Upon Toll-Like Receptor Signaling for Its Cytotoxicity

Blood ◽

10.1182/blood.v116.21.290.290 ◽

2010 ◽

Vol 116 (21) ◽

pp. 290-290

Author(s):

Mahadeo A. Sukhai ◽

Xiaoming Li ◽

Rose Hurren ◽

Xiaoming Wang ◽

Skrtic Marko ◽

...

Keyword(s):

Cell Death ◽

Dendritic Cells ◽

Cell Lines ◽

Leukemia Cells ◽

Ros Generation ◽

Ros Production ◽

Malignant Cells ◽

Toll Like Receptor ◽

Tlr Signaling ◽

Murine Leukemia

Abstract Abstract 290 Known drugs with previously unrecognized anti-cancer activity can be rapidly repurposed for this new indication, given their prior safety and toxicity testing. To identify such compounds, we compiled and screened an in-house library of on-patent and off-patent drugs and screened them to identify agents cytotoxic to hematologic malignancies. From this screen, we identified mefloquine, a quinoline licensed for malaria treatment and prophylaxis. In secondary assays, leukemia and myeloma cell lines were treated with mefloquine for 72 hours and cell viability measured by MTS. Mefloquine decreased the viability of 10/10 human and murine leukemia (LD50 <8.0 μM) and 9/9 human myeloma (LD50 <5.0 μM) cell lines; cell death was confirmed by Annexin V staining. Mefloquine also reduced the viability of 6/6 primary AML samples with LD50 < 5 μ M. These concentrations of mefloquine appear pharmacologically achievable based on prior studies conducted in the context of malaria treatment. In contrast to the effects on malignant cells, mefloquine was significantly less cytotoxic to normal hematopoietic cells (LD50 31.83 ± 5.38 μM) and murine monocyte-derived dendritic cells (LD50 17.56 ± 2.69 μM), Given its in vitro activity, we evaluated the effects of oral mefloquine in mouse xenograft models of leukemia and myeloma. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells, MDAY-D2 murine leukemia cells, or LP1 human myeloma cells, and treated with 50 mg/kg mefloquine, or vehicle alone, by gavage. Oral mefloquine delayed tumor growth by up to 60% in all 4 mouse models without toxicity at doses that appear pharmacologically relevant to humans based on scaling for body surface area. Mefloquine's mechanism of action as an anti-malarial agent is unknown. Therefore, to determine the mechanism by which mefloquine induced cell death in malignant cells, we performed gene expression oligonucleotide array analysis of mefloquine-treated OCI-AML2 cells. At times preceding cell death, mefloquine altered the expression of genes associated with Toll-like receptor (TLR) signaling. For example, we detected 4.5-fold up-regulation of STAT1 and >10-fold up-regulation of its downstream targets, including OAS1, IFIT3 and TRIM22, by 24 hr after treatment. Upregulation of additional TLR targets IRF1, IRF7 and IL-8 was also noted by 8 hours after treatment. Mefloquine also induced early activation of NF-κB with a 2.5± 0.2-fold increase noted after 1 hr, using an ELISA-based DNA binding assay. In contrast to TLR activation in malignant cells, changes in TLR targets were not detected in mefloquine-resistant normal dendritic cells, suggesting that mefloquine's effects on TLR signaling were specific to malignant cells. We next investigated whether TLR activation was functionally important for mefloquine's cytotoxicity in malignant cells. STAT1 activity was required for mefloquine-mediated cell death, as U4A bladder sarcoma cells lacking JAK1 were resistant to mefloquine (LD50 14.6± 4.9 μM), compared to the mefloquine sensitive parental line (LD50 2.3± 0.4 μM). TLR signaling requires the immediate downstream adapter proteins MyD88 and TRIF1. To assess the functional importance of TLR activation for mefloquine induced cell death, we knocked down MyD88 and TRIF1 with siRNA. Double knockdown of MyD88 and TRIF1 completely abrogated mefloquine-induced cell death in K562 leukemia cells at concentrations where control cells exhibited up to 80% loss of viability. TLR signaling and up-regulation of STAT1 can increase reactive oxygen species (ROS) generation. Therefore, we measured ROS generation in leukemia cells after mefloquine treatment. Mefloquine increased ROS production in leukemia cells in a dose-dependent manner within 24 hr. Co-treatment with the ROS scavenger N-Acetyl-L-Cysteine abrogated mefloquine-induced ROS production and cell death. Mefloquine-induced ROS production was also abrogated in MyD88 and TRIF1 double knockdown cells. Our data suggest that the known anti-malarial mefloquine displays preclinical activity in leukemia and myeloma through a mechanism related to TLR activation. Thus, these results highlight TLR activation as a novel therapeutic strategy for the treatment of leukemia and myeloma. Moreover, given its prior toxicology and pharmacology testing, mefloquine could be rapidly advanced into clinical trial for patients with leukemia and myeloma. Disclosures: No relevant conflicts of interest to declare.

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Group I p21-activated kinases in leukemia cell adhesion to fibronectin

10.1101/2020.08.25.266056 ◽

2020 ◽

Author(s):

Kateřina Kuželová ◽

Adam Obr ◽

Pavla Röselová ◽

Dana Grebeňová ◽

Petra Otevřelová ◽

...

Keyword(s):

Cell Death ◽

Cell Adhesion ◽

Cell Lines ◽

Leukemia Cell ◽

Leukemia Cells ◽

Mrna Levels ◽

Comparable Amount ◽

Therapy Targets ◽

Group I ◽

Primary Leukemia

AbstractP21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, including cell adhesion, migration, and apoptosis. PAK function is frequently altered in cancer, and PAK were proposed as therapy targets both in solid tumors and in hematological malignancies. However, current knowledge about PAK function in cell adhesion is mainly based on adherent cell models. Moreover, existing functional differences among the individual PAK family members are unsufficiently characterized.We measured expression of PAK group I members in leukemia cell lines and in primary leukemia cells, both on protein and mRNA levels. In functional assays, we analyzed the effect of two PAK inhibitors with different mechanisms of action, IPA-3 and FRAX597. Changes in cell interaction with fibronectin were monitored through impedance measurement and by interference reflection microscopy. Cytotoxic effects of inhibitors were assessed by Annexin V/propidium iodide test. PAK intracellular localization was analyzed by confocal microscopy.PAK2 transcript was dominant in cell lines, whereas primary leukemia cells also expressed comparable amount of PAK1, which was detected as two transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 transcript levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. The inhibitors had partly opposed effects: IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death. Although many cells accumulated high FRAX597 amounts, low doses were sufficient to kill sensitive cells. FRAX597-induced cell death was fast in the MV4-11 cell line and in primary AML cells.Although PAK group I seem to be essential for leukemia cell adhesion and survival, and might thus serve as therapy targets, many PAK functions still remain to be attributed to individual isoforms and to their functional domains.

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